The T cell repertoire is shaped in the thymus by the selective survival of a limited number of T cell precursors. Two steps: negative and positive selection, ensure that only cells which are l) tolerant to self antigens and 2) MHC restricted, go on to populate the animal. The cells fate is dependent its recognition of self peptide/MHC complexes via the T cell receptor. Model systems to define synthetic Class l MHC binding peptides that induce positive selection of T cells have recently been defined. When present during development, the foreign antigenic peptides cause negative selection, while close variants of this peptide are required for positive selection. The ability of the peptide/MHC ligand to dictate the fate of a T cell is central to the function of the thymus and is the subject of this grant. The primary goal of this work is to define the naturally occurring self peptide/MHC ligands that induce positive selection. The number of distinct self-peptide specificities that contribute to positive selection of a given T cell will first be determined. Secondly, sensitive assays will be developed to facilitate the direct sequencing of one of these peptides by mass spectrometry. The ultimate knowledge of the sequence of a positively selecting ligand will allow us to determine the structural difference between positive and negative selection interactions and to explore the role that these self peptides play in and outside the thymus. The role of peptides in positive selection of the neonatal or germline repertoire will also be examined. The ability to induce positive selection with synthetic peptides is novel. We will capitalize on this property in order to define important aspects of T cell differentiation including: how long ligand engagement is required for and how long thymocytes remain susceptible to positive selection. A further application will be to use peptide induction of positive selection to document the sequence of gene expression events which eventually leads to survival or death. A number of cell death regulatory proteins, including the bcl-2 family members, will be examined. Further studies will focus on the mechanism by which a signal though one receptor can generate such divergent outcomes as positive or negative selection. A biochemical approach will be used to define the proximal signal transduction events that occur in a thymocyte when the T cell receptor engages different ligands. The role of ras activated downstream signaling events in thymocyte differentiation will be addressed using genetic mutants.